It is inappropriate to select insertion sequence (IS) 6110 as the amplification target for the quantitative analysis of Mycobacterium tuberculosis complex (MTC).To develop a novel Taqman real-time polymerase chain reaction (RT-PCR) for the quantitative analysis of MTC by amplifying a single-copy PCR target.Analytic sensitivity and specificity, repeatability and reproducibility, and standard curve were estimated by analysing 18 reference strains and 100 clinical isolates. Diagnostic sensitivity and specificity, positive predictive value (PPV) and negative predictive value (NPV) were evaluated by detecting 50 clinical specimens. Quantitative accuracies of commercial and in-house Taqman RT-PCR were also compared.Analytic sensitivity of this method was 30 colony-forming units/ml and analytic specificity was 100%. In comparison with commercial Taqman RT-PCR (reference), diagnostic sensitivity, diagnostic specificity, PPV and NPV of the novel Taqman RT-PCR were respectively 85.7%, 94.4%, 85.7% and 94.4%. There was no significant difference between the two methods (χ(2) = 0.25, P > 0.05) and there was strong agreement between them (κ = 0.802, P < 0.05). In-house Taqman RT-PCR was more accurate than commercial assay.The novel RT-PCR is sensitive and specific for the detection of MTC and it is also accurate for quantitative analysis of MTC.